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Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer

A technology of Philippine clams and oligopeptides, which is applied in the field of anti-prostate cancer, and the field of preparation of Philippine clams oligopeptides, can solve the problems of less anti-prostate cancer application of Philippine clams active peptides, etc., and can change the cell cycle. , high efficiency, simple process effect

Inactive Publication Date: 2014-12-17
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, with the extension of people's average life expectancy and the improvement of diagnostic technology, the incidence of prostate cancer in my country has shown a significant increase trend, which has seriously affected the quality of life and life expectancy of men over 50 years old in my country, and has become an increasingly important issue in the field of urology. It is an important research topic, but there are few reports about the inhibitory effect of the active extract of Philippine clam on prostate cancer cells, and there are fewer reports about the anti-prostate cancer application of the active peptide of Philippine clam

Method used

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  • Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer
  • Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer
  • Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1: Optimization of enzymolysis conditions

[0056] Screening of protease: wash the fresh Filipino clam, remove the shell and take the meat, then put the clam meat into a homogenizer, pour an appropriate amount of ultra-pure water into the homogenate. Weigh an appropriate amount of homogenized sample, add 2 times the weight of ultrapure water, select trypsin, papain, alkaline protease, and neutral protease as optional enzymes, and use the Fulin-phenol method to determine the enzyme activity (see Yang Yongfang. Study on the preparation technology and anti-prostate cancer activity of enzymatically hydrolyzed polypeptides [D]. Zhoushan: Zhejiang Ocean University, 2011), according to the optimum action temperature and pH value of the proteases shown in Table 1, add 1000u / g protease, enzymatic hydrolysis React for 6 hours. After the reaction is completed, inactivate in a boiling water bath for 15 minutes. After cooling, centrifuge in a pre-cooled centrifuge at 10,0...

Embodiment 2

[0073] Example 2: Preparation and separation and purification of enzymatic oligopeptides

[0074] According to the enzymatic hydrolysis conditions obtained in Example 1, the Philippine clam oligopeptide was prepared, that is, the fresh Philippine clam was washed and then shelled to take the meat, and then the clam meat was put into a homogenizer, and an appropriate amount of distilled water was poured into the homogenate . Weigh an appropriate amount of homogenized sample, add 2 times the weight of water, add 1200u / g trypsin, enzymolysis reaction at pH 8.0, temperature 50°C for 8 hours, inactivate in boiling water bath for 15 minutes after the reaction is completed, and cool Afterwards, centrifuge at 4°C in a pre-cooled centrifuge at 10,000 r / min for 15 minutes, and take the supernatant. Using an ultrafiltration membrane with a molecular weight cut-off of 3KDa, perform ultrafiltration at 20°C for 11 hours to obtain the enzymatic hydrolyzate, which is then freeze-dried for lat...

Embodiment 3

[0077] Example 3: Detection of anti-prostate cancer activity

[0078] Prostate cancer PC-3 and DU-145 cell culture: Prepare 1000ml F12 culture solution, add 1.176g NaHCO3, 100,000 IU of penicillin and streptomycin each, carry out sterile suction filtration, divide into ultra-clean platform and store at -20 Store at low temperature in the refrigerator at ℃, and add 10ml of fetal bovine serum to each 100ml of culture medium before use. Routine culture of human prostate cancer PC-3, DU-145 cell lines, placed in 37 ℃, 5% CO2 incubator culture, 2 to 3 days a passage.

[0079] Cell proliferation inhibition experiment: MTT method was used to detect the survival rate of prostate cancer PC-3 and DU-145 cells cultured in vitro. In the experiment, the PC-3 and DU-145 cell suspensions that had grown to more than 80% and had good morphology, and were in the logarithmic growth phase were inoculated into 96-well plates at 1×104 / mL, 200 μl per well, and cultured for 24 hours. Aspirate and d...

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Abstract

The invention relates to ruditapes philippinarum oligopeptide, the amino acid sequence of which is Asp-Trp-Pro-His. The invention discloses a preparation method of the oligopeptide. According to the preparation method, a target oligopeptide enzymatic hydrolysate is obtained through enzymatic hydrolysis of trypsin; and through ultrafiltration, anion exchange and reversed-phase high-performance liquid chromatography, separation and purification are further carried out to obtain the target oligopeptide. Meanwhile, the invention also discloses an application of the oligopeptide in resisting prostate cancer. Specifically, the oligopeptide can effectively inhibit cell growth of prostate cancer PC-3 and DU-145 cultured in vitro, induce apoptosis and change cell cycle. In comparison with the prior art, the preparation method of the ruditapes philippinarum oligopeptide has advantages of simple technological process, few impurity and high efficiency. Meanwhile, based on the prior art, the invention discloses effectiveness of the oligopeptide in resisting prostate cancer.

Description

technical field [0001] The invention relates to a Philippine clam oligopeptide, in particular to a preparation method of the Philippine clam oligopeptide and its application in the field of anti-prostate cancer. Background technique [0002] Philippine clam (Ruditapes philippinarum) belongs to the mollusk phylum Bivalve clam order clam family, a kind of wide-temperate small bivalve mollusk, commonly known as flower clam in the south, and is listed as the four largest species in my country along with oysters, razor clams and clams. Farmed shellfish is one of the main shellfish in coastal tidal flats and shallow seas. It is delicious, nutritious and high in protein content. [0003] Studies have shown that the extract of Philippine clam has many physiological effects, such as anti-tumor, anti-oxidation, antibacterial and enhancing immunity, etc. In recent years, the anti-tumor research of Philippine clam extract has made great progress, such as Fan Xiuping et al. Food Industry...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P35/00A61K38/07C07K5/113C07K1/36C07K1/20C07K1/34C07K1/18C12P21/06
Inventor 杨最素徐律丁国芳孙瑜黄芳芳郁迪李连军
Owner ZHEJIANG OCEAN UNIV
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